Tacrolimus is an immunosuppressant and a macrolide antibiotic, 100 times more potent than cyclosporine, which selectively inhibits T-lymphocyte activation. It is widely used for the treatment or prevention of rejection in tissue transplantation, autoimmune disorder and infectious diseases caused by pathogenic microorganism. It has an empirical formula of C44H69NO12H2O and the following structure.

Tacrolimus is prepared by the fermentation of Streptomyces tsukubaensis 9993, i.e. monotypic species of Streptomyces, as disclosed in U.S. Pat. Nos. 5,496,727, 5,624,842 and 5,830,717. The fermentation of Streptomyces sp. produces tacrolimus and also provides 17-ethyl derivative (II) known as ascomycin (FK520) and 17-propyl derivative (III) 17-propyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxy-4-azatricyclo-[22.3.1.04.9]octacos-18-ene-2,3,10,16-tetraone). Therefore, there have been various methods reported for separating tacrolimus from a cultured broth.
In U.S. Pat. Nos. 4,929,611 and 5,254,562, a cultured broth filtrate and a mycelia cake extract are combined and eluted on a synthetic adsorption resin (Diaion HP-20), followed by a column chromatography, there providing a highly pure tacrolimus (yield 14%). In more detail, an adsorbed material is washed with distilled water and an organic solvent and elution is carried out. An active fraction is concentrated under reduced pressure and extracted with an organic solvent. The extract is concentrated and column-chromatographied with a normal-phase silica gel (70-230 mesh) by using a mixture of ethylacetate and n-hexane mixture (1:9 v/v, 1:4 v/v, 1:1 v/v and 2:1 v/v) and ethylacetate as mobile phase. Active fractions are collected and concentrated, followed by a column chromatography with a normal-phase silica gel (230-400 mesh) by using a mixture of ethylacetate and n-hexane mixture (1:1 v/v and 2:1 v/v) and ethylacetate as mobile phase. The collected active fraction is subjected to a high-pressure liquid chromatography of a crude product by using the same silica gel (70-230 mesh) and solvent. Elution is conducted by using a column (8?×500 mm) equipped with Lichrosory S160 (Merck Co., Ltd.) as a carrier, and such steps are repeated to provide a highly pure tacrolimus.
In U.S. Pat. Nos. 4,916,138, 4,956,352 and 5,830,717 and European patent publication No. 0,240,773 A1, active fractions concentrated as described in U.S. Pat. No. 4,929,611 are dissolved in acetonitrile, and crystallized at 5° C. to provide a highly pure tacrolimus.
However, this purification process requires the repetition of a high-priced HPLC purification or crystallization after complicated steps using a synthetic adsorbent and a normal-phase resin, while showing a relatively low yield (15%) of tacrolimus.
Although tacrolimus is prepared by using an adsorption and reversed phase resin in U.S. Pat. No. 5,116,756, this method is merely for the identification of a target compound, and cannot be applied for an industry-scaled manufacture.
In the meantime, U.S. Pat. No. 6,492,513 discloses a purification method by using a cation-type ion-exchange resin pretreated with silver salt. According to this purification method, a filtrate of oily compound and a mycelia cake extract were combined and adsorbed onto a synthetic adsorption resin (Diaion HP-20), followed by a column chromatography to provide a crude product. Elution was conducted by developing the crude product with acetone on a synthetic adsorption resin pretreated with silver ion (Capcell Pak SCX UG80™, Shiseido Ltd., Japan). This purification is based on a principle that a cation exchange resin adsorbs tacrolimus more strongly than other impurities due to the formation of silver complex. However, although ascomycin (II) and 17-propyl derivative (III) can be separated, other impurities cannot be separated from tacrolimus by using this process. Therefore, tacrolimus product thus prepared is not suitable for a pharmaceutical purpose due to a low purity.
The information disclosed in the above Background section is only for the enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.